Targeted Drug Regulation on Methylation of p53—BAX Mitochondrial Apoptosis Pathway Affects the Growth of Cholangiocarcinoma Cells

Abstract

To study the mechanism of 5-aza-2-deoxycytidine (DAC; a methylation inhibitor) on growth of the human cholangiocarcinoma QBC939 cell line. A colourimetric assay was used to detect growth of QBC939 cells treated with DAC (0.1-100 μmol/l) over 24 h, 48 h and 72 h. Cell morphology was observed by transmission electron microscopy (TEM). The cell cycle and apoptosis were analysed by flow cytometry. Hypermethylation of the promoters of the p53-BAX mitochondrial apoptosis genes cyclin-dependent kinase inhibitor 2A (CDKN2A), death-associated protein kinase 1 (DAPK1) and PYD and CARD domain containing (PYCARD) was detected by methylation-specific polymerase chain reaction, with and without DAC treatment. DAC inhibited QBC939 cell growth with a half maximal inhibitory concentration of 5 μmol/l at 72 h. After DAC treatment, apoptosis was observed by TEM. Flow cytometric analysis of propidium iodide-positive cells demonstrated increased apoptosis of DAC-treated QBC939 cells (43.04%) compared with untreated cells (4.31%). DAC treatment resulted in demethylation of the gene promoters of CDKN2A and DAPK1 in QBC939 cells. DAC induces apoptosis of QBC939 cells by reactivation of hypermethylated p53-BAX mitchondrial apoptosis genes in cholangiocarcinoma cells.